As mammal neurons do not regenerate if damaged, neurodegenerative diseases such as stroke, Parkinson's disease, or Alzheimer's disease may occur when the neurons are damaged. Accordingly, treatments that can cure diseases due to neuronal death have been actively studied worldwide for a long time, but an adequate treatment has not yet been developed.
Recently, research has been actively carried out to develop a method for treating the neurons damaged by differentiating stem cells, which are pluripotent cells capable of differentiating into various cells, into neurons. As the stein cells can be differentiated into various cells, such method may be fundamental in treating a disease that induces tissue damage. However, as it is not easy to obtain the stem cells or differentiate the stem cells into desired cells, and as the possibility of the stem cells themselves being rejected by patients' immune system can be problematic, the method has not been used universally. However, a cranial nerve disease accompanied by neural damage is considered to be the most appropriate object for the treatment using the stem cells because unlike other tissues, there is little rejection by the immune system in the cranial nervous system tissue, thereby making it possible to expect long-term survival of transplanted cells when the cells are transplanted from outside.
Studies for developing a method for applying the stein cells to treatments of diseases such as stroke, Alzheimer's disease, Parkinson's disease, demyelinating disease, and spinal cord injury are actively under way. For example, International Publication No. WO 2005/003320 discloses a method for inducing the stem cells into neurons, comprising sequentially adding and culturing a basic fibroblast growth factor (bFGF), fibroblast growth factor 8, sonic hedgehog (SHH), and brain-derived neurotrophic factor (BDNF) and ultimately co-culturing with astrocytes. Korean Patent Application No. 10-0495532 discloses a method for differentiating mesenchymal stem cells into neurons by culturing in a culture medium comprising an epidermal growth factor (EGF) and hepatocyte growth factor (HGF) and for proliferating the neurons.
Cholinergic neurons, a type of neuron, are located mainly in the basal forebrain and the hippocampus, having a form in which the neurons extend their axons to other parts of the brain including the cortex. It is known that the cholinergic neurons are directly involved in inducing nervous system diseases in the brain of animals, and that damage to the cholinergic neuron is accompanied by onset of various degenerative cranial nerve diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In particular, learning disabilities, one of the main symptoms of Alzheimer's disease, is predicted to be caused by damage to the cholinergic neurons.
In light of the above, research is actively underway on development of not only a method of using the cholinergic neurons as a therapeutic agent for various degenerative cranial nerve diseases, but also a method capable of differentiating the cholinergic neurons used for treating said diseases from the stem cells. According to what has been reported so far, the embryoid bodies are treated with bFGF, retinoic acid (RA), and SHH to differentiate into the cholinergic neurons. Specifically, the embryoid bodies form early neuroectodermal cells, and are posteriorized when treated with RA and differentiated into the cholinergic neurons when treated with SHH. Additionally, it is known that rat embryonic stem cells can be differentiated into the cholinergic neurons by co-culturing with murine bone marrow origin stromal feeder cells. A method for which the neural progenitor cells are cultured in a culture medium comprising SHH and RA and in a culture medium comprising bfGF and SHH, and in which the culture obtained therefrom is cultured in a culture medium comprising ascorbic acid (AA) and BDNF to differentiate into the cholinergic neurons, is known (Korean Patent Application No. 10-0683199).
However, the cholinergic neurons differentiated by the conventional method include other cells, in addition to pure cholinergic neurons, thereby causing a problem in that the cholinergic neurons cannot be directly used for treating neurodegenerative diseases, and this problem has not yet been resolved.